The presence of insoluble protein aggregates is a pathological hallmark of neurodegenerative disorders such as Parkinson's disease (PD), dementia with Lewy bodies (DLB), Alzheimer's disease, Huntington's disease and prion protein disorders. In synucleinopathies such as PD and DLB, the Lewy body is the pathological protein aggregate that is found in the cytoplasm of susceptible neuronal populations. Lewy bodies are eosinophilic inclusions containing numerous aggregated proteins, including alpha-synuclein (1syn), which suggests a causative role for this protein in these disorders. Misfolding and aggregation of 1syn are thought to be critical cofactors linked to disease development. In particular, the accumulation of nonfibrillar dimers and oligomers of 1syn, which serve as intermediates for fibrillar Lewy body formation, is thought to cause neurodegeneration. Because oligomeric forms of 1syn are believed toxic to cells, an important therapeutic strategy will be to reduce oligomer formation or prevent it altogether. In this application we propose to study the role of oligomeric 1syn in synucleinopathies and examine how specific modifications affect toxicity and oligomer formation. To enable the direct study of 1syn oligomerization, we have applied novel strategies including protein complementation assays (PCAs), size exclusion chromatography (SEC) and fluorescence correlation spectroscopy (FCS) by which to identify and follow 1syn oligomer formation, thus providing a powerful toolbox of techniques with which to study manipulations affecting 1syn-induced toxicity. This application is divided into three coordinated and related aims that will characterize 1syn oligomers and investigate the role of 1syn oligomers in disease pathogenesis with a view to determining the effect of disease- related modifications and molecular chaperones on 1syn oligomer formation and subcellular location (aim 1 & 2). We also propose to recapitulate 1syn oligomerization in vivo via stereotactic injection of recombinant adeno-associated virus and examine the resulting toxicity and subcellular localization of 1syn oligomers in vivo, as well as the effect of molecular chaperone interventions identified in aim 2 (aim 3). We propose that this application offers an effective strategy by which to identify and modulate 1syn oligomer formation, and targets therapeutics to a pre-toxic stage of fibrillization, where rescue of the soluble, monomeric forms of 1syn can substantially impact disease progression. If successful, the experiments described will significantly advance the study of 1syn-induced toxicity and related oligomer formation, and contribute to the development of successful therapeutic strategies to treat PD and related disorders. Thus, this study is in accordance with the mission of NINDS - to reduce the burden of neurological disease. PUBLIC HEALTH RELEVANCE: A common feature of neurodegenerative diseases is the excessive accumulation of misfolded proteins and a disruption in cellular function. There is strong evidence to suggest that alpha-synuclein (1syn) accumulation is an early step in the development of both sporadic and inherited Parkinson disease (PD). Because 1syn accumulates and aggregates in neurons, we hypothesize that a complete understanding of 1syn misfolding, oligomerization and aggregation will be crucial to the development of new therapeutic strategies for the treatment of PD and related synucleinopathies. The goals of the current application are to understand 1syn oligomer formation and how it relates to disease development in humans and to further explore factors that modify and rescue 1syn aggregation.